The long-term aim of this work is to understand the underlying molecular mechanisms by which naturally occurring stimuli and inhibitors regulate the opening and closing of the BK-type calcium (Ca2+)-activated potassium (K+) channel. A common characteristic shared by BK channels with essentially all other ion channels is that sensing of a physiological stimulus on one part of the protein is coupled to regulation of a key functional property occurring on another part of that protein. In the case of BK channels, sensing of either changes in membrane voltage or changes in cytosolic Ca2+ regulate the activation of ion flux through the channel. That BK channels respond independently to two distinct physiological signals is an advantage for investigation of the underlying molecular steps that link these processes. Understanding these processes is important for two reasons. First, because of the important role of BK channels in a number of physiological systems, understanding regulation of BK channels promises to provide insights into a number of different disease pathologies and provide strategies for diseases amelioration. Second, by taking advantage of unique features of the BK channel, the work will provide new general insights regarding the mechanisms of regulation of ion channels. Such insights promise to be of utility in understanding regulation of essentially all ion channels and other proteins. This project focuses on two major aspects of how activation of BK channels is regulated. First, the role of the BK channel pore-lining S6 helix in defining channel gating and coupling to gate opening will be examined. The BK S6 helix has been shown to be unique among K+ channels in terms of the residues that face the aqueous inner pore. As a corollary, BK S6 residues participate in unique state-dependent interactions favoring either open or closed conformations. We hypothesize that defining these interactions will be critical for understanding the BK channel machinery. Using methods of electrophysiology combined with molecular biology, we will probe the interactions of BK S6 residues with other parts of the channel and examine state- dependent movements of the S6 residues. Second, BK channels are inhibited by a family of tremorogenic toxins. These toxins are useful probes of channel conformation and investigation of the mechanism of action of these toxins promises to provide novel insight in a new mechanism of channel regulation. These toxins selectively stabilize closed channel states. Investigation of this category of inhibitory mechanism is expected to have broad significance for inhibition of a number of other ion channels. The understanding of regulation of BK channel function to be achieved in this work is of potential medical importance, not only because BK channels are promising therapeutic targets in asthma, epilepsy, tumor growth, and ischemic insults, but also because pathological alterations of BK channels may underlie various disease states.